The efficiency of chemotherapeutic treatment of tumors is hindered by the onset of multi-drug resistance (MDR) resulting from the overexpression of certain membrane-bound proteins in cancer cells. These membrane-bound MDR proteins include the MDR1 protein (alternatively termed P-glycoprotein), MRPs, and their functional analogs, breast cancer resistance protein (BCRP) and others. Cytostatic agents, e.g., Vinca alkaloids, anthracycline derivatives and other clinically effective anti-cancer agents are actively transported out of cancer cells by these MDR proteins, thereby decreasing their efficacy.
These same transport proteins are expressed in normal cells including the epithelial cells of the intestine, liver, kidney and placenta, and brain endothelial cells that form the blood-brain barrier (BBB). The presence of MDR transport proteins in these normal cells can have adverse effects on the absorption, distribution and elimination properties of many drugs and xenobiotics.
Cell permeable, hydrophobic ester derivatives of some fluorescent dyes are also actively exported from cells by MDR proteins. However, when the ester derivatives reach the cytosol, intracellular esterases cleave the esters in the fluorescent dyes, and the MDR1 proteins fail to export the resulting free dye compounds (Homolya, et al. (1993) J. Biol. Chem. 268:21493-96). It has been demonstrated that the commercially available compound, calcein-AM (calcein-acetoxy-methylester) is, unlike free calcein, an activator of the MDR-dependent ATPase. It has also been shown that calcein accumulation in the cell following calcein-AM uptake is reduced by the presence of MDR1 activity. Likewise, other MDR proteins such as the MRPs export free calcein from cells. This activity of MRP proteins decreases cellular levels of calcein, thereby decreasing fluorescence. Thus, expression of either MDR1 or MRPs leads to decreased cellular fluorescence.
It has been demonstrated that calcein-AM is useful for the functional analysis of the presence of multi-drug resistance in cells (Hollo, et al. (1994) Biochim. Biophys. Acta. 1191(2):384-8; U.S. Pat. Nos. 6,391,656 and 6,277,655; WO 96/06945). Cells overexpressing MDR proteins export the penetrating calcein-AM molecules via an active transport mechanism, and thus the rate of transformation of calcein-AM to fluorescent calcein (or other fluorescent calcein derivatives) and the rate of accumulation of the fluorescent product(s) within the cells are significantly reduced relative to wild-type cells.
Commercially available fluorescent dye-based kits such the MULTIDRUG RESISTANCE DIRECT DYE EFFLUX ASSAY™ available from Chemicon International, Inc. (Temecula, Calif.), the MULTIDRUGQUANT™ Assay Kit available from Solvo Biotechnology (Budapest, Hungary) and the VYBRANT™ Multidrug Resistance Assay Kit available from INVITROGEN™-MOLECULAR PROBES™ (Carlsbad, Calif.) can be used for detecting MDR protein activity, however, these assays generally require washing steps to remove extracellular solutions. Therefore, these assays are time consuming and generally do not provide real-time MDR protein activity, thus providing limited data.
Drug resistance profiling is also of interest in the clinical setting to determine substrate specificity and drug efflux activity of the various multi-drug resistance proteins in a given tumor sample. Therefore, there is a need in the art for a rapid, highly sensitive, reproducible in vitro multi-drug resistance assay to demonstrate transport activity of various multi-drug resistance proteins (e.g., MDR1, MRP1, cMOAT) in the plasma membrane. The present invention meets this need in the art.